The present study was conducted on 81 commercial broiler chicken farms aged (1-45 days) located in five different centers at Qena province (Qena, Qift, Deshna, Qus and Nag-Hammadi), For bacteriological examination, Samples were taken from diseased and freshly dead chickens (liver; lung; intestine; trachea; nasal cavity; kidneys; yolk sacs; pericardium; and air sacs). The results revealed that 50 out of 600 samples (8.3%) were positive for E. coli isolation, where 30 isolates (9.0%) from 332 baby chicks (up to one week), 20 isolates (7.4%) from 268 broilers (from one week to 6 weeks) were positive for E. coli isolation. Thirty-seven seogroups of E. coli were obtained by serological identification (O1, O6, O8, O20, O26, O27, O44, O86a, O115, O125, O128, O136, O146, O152, O158, O159, O164, O166, O167,O168 and O169) represented as 4 strains were serotyped O27 (8%), 3 strains O44 (6%), 3 strains O125 (6%), 3 strains O152 (6%), 3 strains O159 (6%), 2 strains O1 (4%), 2 strains O6 (4%), 2 strains O20 (4%), 2 strains O26 (4%), 2 strains O166 (4%), 1 strain for each of O8, O86a, O115, O128, O136 ,O146, O158, O164, O167, O168, O169 with (2%) for each of them and 13 isolates were untyped (26%) by the available antisera. Multiplex PCR showed that ompA virulence gene was detected in all serogroups except O8. Moreover, papC virulence gene was detected in all serogroups except O8, O86a, O136, O146, O167 and O169. While Stx2 virulence gene was detected only in serotypes O27 & O125. The eaeA gene was amplified in serogroups O1, O20, O26, O115, O152, O166, O168 and O169. Finally, tsh virulence gene was detected in all serogroups except O20, O26, O86a, O115, O128, O167 and O169.